The long-term goal of our research is to develop potent and safe anthrax vaccines and to understand the fundamental mechanisms of anthrax toxicity and the response of the immune system to anthrax infection. The aim of this proposal is to explore enhancement of the immunogenicity of protective antigen (PA), the major antigen of currently available anthrax vaccine. PA is the central component of the tripartite anthrax toxin complex, responsible for binding to cells and transporting lethal factor (LF) and edema factor (EF) into the cytosol where intoxication occurs. This pathway has presumably evolved to be optimal for the targeted delivery of LF/EF by PA to kill the host while minimizing the induction of host immunity. Based on this assumption, we hypothesize that the immunogenicity of PA can be augmented by engineering the protein such that it will not only block intoxication steps but also change its cellular trafficking. Such modified PA would be expected to possess much improved immunogenicity and may induce significant amounts of protective antibodies. In support of this hypothesis, we recently demonstrated that a dominant-negative inhibitory (DNI) mutant is more potent than PA in inducing protective antibodies. In the present study, we will compare the immunogenicities of native PA and four distinct classes of PA mutants (Aim 1). These mutants, each being impaired in a different step of PA-mediated intoxication, provide valuable molecular tools for probing the sequence of cellular and biochemical pathways that may lead to enhanced immunogenicity of mutant vs. native PA. To dissect the basic mechanisms of PA immunogenicity and to explain the enhanced antibody response to DNI, we recently proposed that DNI, which is defective in inserting into endosomal membranes and does not translocated LF/EF, may remain trapped endosomally and is, therefore, processed more efficiently by antigen-presenting cells than native PA. To experimentally test this "endosomal trapping" hypothesis, we will investigate whether DNI enhances the immunogenicity of co-delivered antigens such as LF, LFn-GFP, and LFn-PGA since DNI is expected to also trap these antigens within endosomes (Aim 2). Furthermore, we will examine the cellular fates of PA and DNI in greater detail (Aim 3). This study is highly significant in that it will not only shed light on the basic mechanisms of PA immunogenicity and the basic biology of anthrax intoxication but it will also yield more potent and safer anthrax vaccine candidates.